A pH indicator agar plate method was used to screen for esterase activities for hydrolysis of 2-ethylhexyl butyrate. Seven hundred and fifty-seven selected microbial cultures, including 325 bacteria, and 432 yeasts and actinomycetes from the ARS Culture, Collection, were screened. Among them, 62 cultures hydrolyzed 2-ethylhexyl butyrate. Of these strains only 17 showed lipase activity on a rhodamine B lipase screen. The reaction products, 2-ethyl-1-hexanol andn-butyric acid were confirmed by gas-liquid chromatography (GC) and GC/MS analyses. The yield of 2-ethyl-1-hexanol varied depending on the strains of the microorganisms, with the highest yield at 79.1% by a strain ofPseudomonas myxogenes Product analyses with a cyclodextrin GC chiral column showed that two strains ofPseudomonas produced, greater than 80% enantiomeric excess of S(+)-2-ethyl-1-hexanol.